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• • A—HUMAN NECESSITIES
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  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
  • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • C07K—PEPTIDES
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/0331—Animal model for proliferative diseases
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/035—Animal model for multifactorial diseases
  • A01K2267/0381—Animal model for diseases of the hematopoietic system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• the invention relates to transgenic non-human mammals that reproduce the human pathology that has its origin in stem cells using as a strategy the expression of the genes involved in said pathology in human beings by means of a promoter that directs the expression of a transgene in Sca-cells. 1 + .
• Transgenic animals are animals that carry an exogenous gene (transgene) in their genome, which has been introduced into the germ cells of the animal, or in an ancestor thereof, at an early stage of development.
• the introduction of a transgene in an animal can have the purpose of studying the behavior, expression or function of the introduced gene. Alternatively, it can pursue a genetic improvement in the affected individual for therapeutic or animal improvement purposes.
• transgenic mammals The generation of transgenic mammals is well established [see, for example, Hogan, Constantini & Lacy (1986), "Manipulating the Mouse Embryo. A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor (1986)] and proof of this It is the high number of articles and patents that describe transgenic mammals.
• US patents US 4,736,866, US 4,873,191, US 5,175,383 and US 5,175,384, among others, may be cited.
• transgenic mammals are valuable models for the in vivo study of compounds that could potentially be useful in the treatment or prevention of said disease and / or in the development of useful methods for the diagnosis of said disease.
• human pathology that has its origin in stem cells includes a group of human diseases, both neoplastic and non-neoplastic, which they originate in stem cells, both hematopoietic and non-hematopoietic, for example, myeloid leukemias, lymphoid leukemia of line B, lymphoid leukemia of lineage T, lymphomas, sarcomas and pathology of stem cell development, for example, congenital immunodeficiencies, anemia of Fanconi, etc.
• Neoplastic malignant pathology (whose almost all originates from stem cells) is currently treated in humans through a combination of chemotherapy and radiotherapy and / or surgery strategies, strategies that do not discriminate between normal and tumor cells.
• BCR-ABL p210 acute lymphoblastic leukemia of line B
• BCR-ABL p190 acute lymphoblastic leukemia of line T
• HOX11, RHOM2 / LMO-2 and TAL1 acute lymphoblastic leukemia of line T
• the invention faces the problem of developing animal models that reproduce the human pathology that has its origin in stem cells.
• transgenic mice that contain a DNA construct comprising a gene that is created and / or activated by chromosomal abnormalities associated with different types of leukemia, or by stem cell migration hematopoietic or embryonic, said gene being under the control of a promoter that directs the expression of said gene in Sca-1 + cells, such as stem cells, develop varying levels of human pathology, both neoplastic and non-neoplastic, originating in cells hematopoietic or non-hematopoietic stem.
• transgenic mammals By directing the expression of the different genes to the stem cell compartment through the use of a promoter that directs the expression of such genes in Sca-1 cells, a set of animal models that reproduce human pathology has been generated. This fact has been demonstrated by generating a set of transgenic mice that have genotypes that confer a greater tendency to develop human pathology originating in stem cells when compared to non-transgenic mice.
• the transgenic mammals provided by this invention therefore constitute a new and useful model for the study of said diseases and for the evaluation of compounds useful for the treatment and / or prevention of said diseases.
• the animal models that reproduce the human pathology originating in stem cells allow: a) to have a unique tool to study how this pathology is generated, maintained and developed; b) predict the efficacy of potentially valid therapies for humans; c) discover new therapies, and d) make genomic identifications of alleles that suppress or increase the natural evolution of each pathology.
• an object of this invention is a DNA construct comprising a gene that is created and / or activated by a chromosomal abnormality associated with a human pathology originating in stem cells, said gene being under the control of a promoter that directs the expression of said gene in Sca-1 cells.
• a further object of this invention is a non-human mammal.
• transgenic that has a genotype that confers a greater tendency to the development of human pathology with stem cell origin when compared to that of a non-transgenic mammal.
• Said transgenic non-human mammal is useful, among other purposes, for studying said pathology and evaluating compounds potentially useful for treating and / or preventing said pathology. Therefore, an object of this invention is a transgenic non-human mammal that contains a transgene and its progeny.
• transgenic mouse that contains a transgene and its progeny.
• said transgenic mouse is selected from the graph formed by Sca-l + BCR-ABL p21 °, Sca-l + BCR-ABL pl90 3 Sca-1 + Slug, Sca-1 + Snail, Sca-1 + HOXl 1, Sca-l + RHOM2 / LMO-2, Sca-1 + TALl.
• Another additional object of this invention is a process for the preparation of a transgenic non-human mammal useful as an animal model for the in vivo study of human pathology with stem cell origin.
• Another additional object of this invention is a transgenic non-human mammalian cell line that contains said DNA construct in its genome.
• Another additional object of this invention is the use of a promoter that directs the expression of a gene in Sca-1 + cells for the generation of animal models that reproduce human pathology originating in stem cells.
• Another additional object of this invention is the use of said transgenic non-human mammal in the evaluation of compounds potentially useful for the treatment and / or prevention of human pathology originating in stem cells.
• Another additional object of this invention is the use of a promoter that directs the expression of a gene in Sca-1 + cells as a vehicle for therapeutic strategies.
• Pharmaceutical compositions containing a DNA construct comprising said promoter and a therapeutic gene, as well as the use of said promoter that directs the expression of a gene in Sca-1 + cells constitute additional objects of this invention.
• Figure 1 consists of a set of graphs and photographs that constitute the phenotypic and histological demonstration of Sca-l + BCR-ABL p210 mice with chronic myeloid leukemia.
• Figure 1A shows the representative analysis of the cellular composition present in the bone marrow (BM) and in the peripheral blood (PB) of Scal + BCR-ABL 21 ° mice.
• Cells isolated from Sca-l + BCR-ABL p21 ° mice were stained with the indicated monoclonal antibodies [Gr-1, for the granulocytic series; Macl for the myelomonocytic series and Seal for the stem series] and were analyzed by flow cytometry. The percentage of positive cells is indicated.
• FIG. 1B shows the result of the representative histological examination of spleen, liver and lymph node sections of Sca-l + BCR-ABL 210 mice with chronic myeloid leukemia. All sections were stained with hematosilin and eosin. The arrows indicate the presence of fibrosis and megakaryocytes typical of the chronic myeloid leukemia process.
• Figure 1C shows a representative staining of the peripheral blood of Sca-l + BCR-ABL p210 mice stained with Gie sa.
• Figure 2 consists of a set of graphs and photographs that constitute the phenotypic and histological demonstration of myeloid and lymphoid blast crisis in Scal + BCR-ABL p210 mice with chronic myeloid leukemia.
• Figure 2A shows the representative analysis of the cellular composition present in the bone marrow (BM), spleen and peripheral blood (PB) of Sca-l + BCR-ABL 210 mice in blast crisis.
• BM bone marrow
• PB peripheral blood
• Figure 2C shows a representative staining of the peripheral blood of Sca-l + BCR-ABL p210 mice in blast crisis stained with Giemsa where blast cells are seen.
• Figure 3 consists of a set of graphs and photographs that constitute the phenotypic and histological demonstration of Sca-l + BCR-ABL p190 mice with acute lymphoblastic line leukemia B.
• Figure 3A shows DNA analysis by Southern blot of Sca mice -l + BCR-ABL 190 and hybridized controls with a specific ABL probe. The presence of the transgene (pl90) is observed in Sca-1 + BCR-ABL P mice.
• Figure 3B shows the results of the representative histological examination of spleen, liver, lung and peripheral blood stains of control mice and Sca-l + BCR-ABL pl9 °. All histological sections were stained with hematosilin and eosin and peripheral blood stains with Giemsa and evidenced the presence of leukemic cells in Sca-l + BCR-ABL p190 mice .
• Figure 3C shows the representative analysis of the cellular composition present in the bone marrow (BM) and peripheral blood (PB) of control mice and Sca-l ⁇ BCR-ABL p190 mice .
• BM bone marrow
• PB peripheral blood
• the invention provides a DNA construct, hereinafter referred to as the DNA construct of the invention, comprising a gene that is created and / or activated by a chromosomal abnormality associated with a human pathology originating in stem cells, said gene being under the control of a promoter that directs the expression of said gene in Sca-1 + cells.
• said chromosomal abnormality associated with a human pathology originating in stem cells is selected from the chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of strain B, acute lymphoblastic leukemia of strain T, or with the Hematopoietic or embryonic stem cell migration.
• the stem cell factor are an example of human pathology associated with the migration of hematopoietic or embryonic stem cells.
• the expression "gene that is created and / or activated by a chromosomal abnormality associated with a human pathology originating in stem cells", hereinafter, activatable gene refers to a gene or gene fusion that when incorporated into the genome of a mammal, the probability that said mammal develops the pathology to which said gene or gene fusion is associated increases.
• said activatable gene is a gene that is created and / or activated by a chromosomal abnormality associated with a human pathology originating in stem cells selected from the chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of lineage. B, acute lymphoblastic leukemia of strain T, or with the migration of hematopoietic or embryonic stem cells.
• said activatable gene is selected from the genes identified as BCR-ABL p210 , BCR-ABL p190 , Slug, Snail, HOX11, RHOM2 / LMO-2 and TAL1.
• said activatable gene is selected from the following genes: human BCR-ABL p210 , gene fusion that occurs as a consequence of t (9; 22) (q34; ql 1) and is associated to chronic myeloid leukemia; the patients who present this chromosomal anomaly develop over time a blastic crisis, which is an evolutionary phenomenon characteristic of said disease;
• BCR-ABL p190 human an oncogene generated by the chromosomal translocation t (9; 22) and associated with acute lymphoblastic leukemia of line B;
• Murine Slug a gene that participates in the mobilization of hematopoietic stem cells
• Murine Snail a gene from the Slug family that participates in the migration of embryonic stem cells
• Human HOX11 a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of T-line;
• Human RHOM2 / LMO-2 a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of T-strain; Y
• TAL1 murine a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of strain T.
• Activatable genes identified as BCR-ABL p21 °, BCR-ABL p190 , HOX11, RHOM2 / LMO-2 and TAL1 are described in Annu. Rev. Genet. (1997) 31: 429-453;
• the Slug gene has been described by Nieto MA, Sargent MG, Wilkinson DG and Cooke J (1994) "Control of cell behavior during vertébrate development by Slug, a zinc-fmger gene".
• the promoter that directs the expression of the activatable gene in Sca-1 + cells is a nucleic acid sequence involved, and necessary, at the start of transcription, which directs the expression of the activatable gene in Sca-1 cells, and includes the site binding of RNA polymerase.
• the term "promoter" may include other sites at which transcriptional regulatory proteins can bind.
• the promoter that directs the expression of the activatable gene in Sca-1 + cells is the mouse pLy-6E.l promoter or a functional fragment thereof, that is, capable of directing tissue specific expression of the Different transgenes in mice.
• the pLy-6E.l promoter is well characterized and contains all the elements necessary for selective expression in Sca-1 cells [Miles C, Sánchez MJ, Sinclair A, and Dzierzak, E. (1997) "Expression of the Ly-6E .l (Sca-1) transgene in adult hematopoietic stem cells and the developing mouse embryo ". Development 124: 537-547].
• the term "operably linked” refers to the orientation of the promoter with respect to the sequence of the activatable gene. The promoter is placed in such a way that it is capable of controlling or regulating the expression of said activatable gene.
• the DNA construct of the invention can be easily obtained by conventional methods of digestion with restriction and binding enzymes, and the like such as those described by Sambrook, Fitsch and Maniatis, eds., (1989) "Molecular Cloning: A Laboratory Manual”. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY.
• the DNA construct of the invention can be used, if desired, for the production of vectors useful for transforming mammalian embryos and generating transgenic animals using conventional methods such as those described by Sambrook et al., Cited supra.
• the DNA construct of the invention can be used to obtain a linear DNA fragment useful for microinjection of DNA to fertilized oocytes in order to generate transgenic animals.
• Said linear DNA fragment useful for microinjection can be obtained by cutting with restriction enzymes in order to obtain a linear DNA fragment comprising the activatable gene.
• the invention provides a transgenic non-human mammal that contains in its genome a DNA construct of the invention, that is, a constnition comprising a gene that is created and / or activated by a chromosomal abnormality associated with a human pathology.
• a chromosomal abnormality associated with a human pathology originating in stem cells, for example, chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of line B, acute lymphoblastic leukemia of line T, or with the migration of hematopoietic or embryonic stem cells, said gene being under the control of a promoter that directs the expression of said gene in Sca-1 + cells.
• the transgenic non-human mammal provided by this invention consequently possesses a genotype that confers a greater tendency to the development of human pathology originating in stem cells when compared to that of a non-transgenic mammal.
• Said transgenic non-human mammal is useful, among other purposes, for studying said pathology and evaluating compounds potentially useful for treating and / or preventing said pathology.
• non-human mammal includes any non-human animal belonging to the class of mammals, for example, mice.
• the transgenic non-human animal provided by this invention is a transgenic mouse identified as: Sca-l + BCR-ABL p210 : these mice develop chronic myeloid leukemia;
• Sca-1 + Slug these mice mobilize hematopoietic stem cells
• Sca-1 + Snail these mice mobilize stem embryonic cells
• Sca-1 + HOXll these mice develop acute lymphoblastic leukemia of T-strain
• the DNA construct of the invention has been introduced into said mammal, or an ancestor thereof, into an embryogenic stage, for example, in the stage of a cell, or fertilized oocyte, and, generally, not later than the 8-cell stage.
• the invention provides a method for the preparation of a transgenic non-human mammal that has a chromosomal abnormality associated with a human pathology originating in stem cells, comprising
• said chromosomal abnormality associated with a human pathology originating in stem cells is a human pathology selected from the chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of strain B, acute lymphoblastic leukemia of strain T, or with the migration of hematopoietic or embryonic stem cells (for example, alterations of the c-kit receptor or its ligand (SCF)), in which case, the offspring is analyzed to assess the existence of genes activated and / or created by the Chromosomal abnormality associated with human pathology originating in stem cells in question.
• SCF c-kit receptor or its ligand
• Non-human transgenic of the present invention One method is to transfect the embryo with said nucleic acid sequence as it occurs naturally, and select the transgenic animals in which said sequence has been integrated into the chromosome in a locus that results in the activation of said sequence. Another method involves modifying the nucleic acid sequence, or its control sequences, before introducing it into the embryo. Another method is to transfect the embryo using a vector that contains the nucleic acid sequence to be introduced.
• the introduction of the DNA construct of the invention into the germ line of a non-human mammal is performed by microinjection of a linear DNA fragment comprising the activatable gene operably linked to the promoter that directs expression in Sca cells. -1 + in fertilized oocytes of non-human mammal.
• the fertilized oocytes can be isolated by conventional methods, for example, causing ovulation of the female, either in response to intercourse with a male or by induction by treatment with luteinizing honnone. In general, superovulation is induced in females by hormonal action and they cross with males. After For an appropriate period of time, females are sacrificed to isolate fertilized oocytes from their oviducts, which are maintained in an appropriate culture medium. Fertilized oocytes can be recognized under the microscope by the presence of pronuclei. The microinjection of the linear DNA fragment is advantageously carried out in the male pronucleus.
• the fertilized oocytes After the introduction of the linear DNA fragment comprising the DNA construct of the invention in the fertilized oocytes, these are incubated in vitro for an appropriate period of time or reimplanted in pseudopregnated mothers (obtained by copulating females with sterile males) . Implantation is performed by conventional methods, for example, anesthetizing the females and inserting a sufficient number of embryos, for example, 10-20 embryos, into the oviducts of the pseudopregnated mothers. After the pregnancy, some embryos will carry out the pregnancy and will lead to transgenic non-human mammals, which theoretically must carry the DNA restriction of the invention integrated in their genomes and present in all cells of the organism. This progeny is the G0 generation and its individuals are the "transgenic founders".
• Confirmation that an individual has incorporated the injected nucleic acid and is transgenic is obtained by analyzing the individuals of the progeny.
• DNA is extracted from each individual and is analyzed by conventional methods, for example, by polymerase chain reaction (PCR) using specific primers or by Southern blot or Northern blot analysis using, for example, a probe that is complementary to at least a part of the transgene, or by Western blot analysis using an antibody against the protein encoded by the transgene.
• PCR polymerase chain reaction
• Southern blot or Northern blot analysis using, for example, a probe that is complementary to at least a part of the transgene, or by Western blot analysis using an antibody against the protein encoded by the transgene.
• Other methods for assessing the presence of the transgene include, without limitation, appropriate biochemical assays, such as enzymatic and / or immunological assays, histological stains for particular markers, enzymatic activities, etc.
• the inserted transgene is transmitted as a Mendelian character so it is not difficult to establish stable lines of such an individual. If the individuals of the G0 intersect with the paternal strain (backcrossing) and the transgene It behaves like a Mendelian character, 50% of the progeny will be heterozygous for the inserted transgene (hemizigotic). These individuals constitute the Gl progeny and a transgenic line that can be maintained indefinitely by pairing hemizigotic Gl with normal individuals. Alternatively, Gl individuals can cross each other to produce 25% homozygous for the inserted transgene, 50% hemizigotics and 25% without transgene as long as the transgene does not affect the viability of the offspring.
• the progeny of a transgenic non-human mammal provided by this invention can therefore be obtained by coupling the transgenic animal with an appropriate individual, or by in vitro fertilization of eggs and / or sperm of transgenic animals.
• the term "progeny” or “progeny of a transgenic non-human mammal” refers to each and every descendant of each generation subsequent to that of the originally transfonted non-human mammals. The progeny can be analyzed to detect the presence of the transgene by any of the previously mentioned methods.
• the invention also relates to a transgenic non-human mammalian cell line that contains in its genome a DNA construct of the invention, that is, a construct comprising a gene that is created and / or activated by a chromosomal abnormality associated with a human pathology with stem cell origin, for example, chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of line B, acute lymphoblastic leukemia of line T, or with the migration of hematopoietic or embryonic stem cells, said gene being under the control of a promoter that directs the expression of said gene in Sca-1 + cells.
• said cell line is a murine cell line.
• the invention relates to the use of a promoter that directs the expression of a gene in Sca-1 + cells for the generation of non-human animal models that reproduce human, neoplastic or non-neoplastic pathology, which has its origin in stem, hematopoietic or non-hematopoietic cells, such as non-human animal models presenting a chromosomal abnormality associated with a human pathology originating in stem cells, for example, a chromosomal abnormality associated with chronic myeloid leukemia, acute lymphoblastic leukemia of strain B, acute lymphoblastic leukemia of strain T, or the migration of hematopoietic or embryonic stem cells.
• said promoter that directs the expression of a gene in Sca-1 cells is the mouse pLy-6E.l promoter.
• the transgenic non-human mammal provided by this invention, its progeny or the cell line provided by this invention, are useful for, among other applications, evaluating compounds potentially useful for treating and / or preventing a chromosomal abnormality associated with human, neoplastic or non-human pathology.
• the invention also relates to the use of said transgenic non-human mammal, its progeny or a cell line provided by this invention, in the evaluation of compounds potentially useful for treating and / or preventing a chromosomal abnormality associated with human pathology that has its origin in stem cells.
• said chromosomal abnormality is selected from the chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of strain B, acute lymphoblastic leukemia of strain T, or the migration of hematopoietic or embryonic stem cells.
• the evaluation of the compound potentially useful for the treatment and / or prevention of said human pathology originating in stem cells can be performed by administering the compound to be tested to said transgenic animal, at different dosages, and evaluate the physiological response of the animal over time.
• the administration of the compound to be tested may be orally or parenterally depending on the chemical nature of the compound to be evaluated. In some cases it may be appropriate to administer the compound in question together with co-factors that enhance the efficacy of the compound.
• the evaluation of the compound potentially useful for the treatment and / or prevention of said human pathology originating in stem cells can be performed by adding the compound to be tested to a medium of cell culture, for an appropriate period of time, at different concentrations, and evaluate the cellular response to the compound over time using the appropriate biochemical and / or histological assays. Sometimes it may be appropriate to add the compound in question to the cell culture medium together with co-factors that enhance the efficacy of the compound.
• the invention relates to the use of a promoter that directs the expression of a gene in Sca-1 cells as a vehicle of therapeutic strategies for the treatment and / or prevention of a chromosomal abnormality associated with a human pathology that has its origin in stem cells, for example, in the treatment and / or prevention of a chromosomal alteration associated with a human pathology originating in stem cells selected from the chromosomal abnormalities associated with chronic myeloid leukemia, the acute lymphoblastic leukemia of strain B, the acute lymphoblastic leukemia of T-line, or with the migration of hematopoietic or embryonic stem cells.
• the promoter directs the expression of a transgene in Sca-1 + cells, such as stem cells, allows the specific and exclusive expression of different therapeutic strategies in stem cells.
• said promoter that directs the expression of a gene in Sca-1 + cells is the mouse pLy-6E.l promoter.
• therapeutic strategies for the treatment and / or prevention of chromosomal abnormalities associated with human pathologies that have their origin in stem cells developed through the use of a promoter that directs the expression of a gene in Sca-1 + cells as a vehicle for said therapeutic strategies are the inactivation of a gene or malignant gene fusion, for example, in neoplasms, or the inclusion of a functional gene that is lacking or the replacement of a defective gene by a functional gene, for example, in immunodeficiencies
• These therapeutic strategies can be materialized by (i) the preparation of a DNA restriction comprising said promoter that directs the expression of a gene in Sca-1 + cells and a therapeutic gene suitable for the treatment and / or prevention of the anomaly chromosome associated with human pathology originating in stem cells to be treated and / or prevented, said therapeutic gene being under the control of said gene that directs expression in Sca-1 cells; and (ii) the incorporation of said DNA construct into a vector or system that helps the process of transferring an exogenous
• said vectors or systems may be viral vectors, for example, based on adeno virus, lentivirus, retro virus, etc., or non-viral such as DNA-liposome, DNA-polymer, DNA-polymer-liposome complexes, etc. [see “Nonviral Vectors for Gene Therapy”, edited by Huang, Hung and Wagner, Academic Press (1999)].
• the term "therapeutic gene” refers to a gene or gene construct useful for the treatment and / or prevention of a chromosomal abnormality associated with human pathology originating in stem cells, and includes ribozymes, genes , antisense gene constructs or fusions, and, in general, any gene, fusion or gene construct useful against the gene created and / or activated by the chromosomal abnormality in question, for example, chronic myeloid leukemia, acute lymphoblastic leukemia of strain B, leukemia acute lymphoblastic strain T, alterations of the c-kit receptor or its ligand (SCF), etc.
• SCF c-kit receptor or its ligand
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a DNA construct comprising a therapeutic gene suitable for the treatment and / or prevention of a chromosomal abnormality associated with human pathology originating from stem cells to be treated and / or prevented.
• a promoter that directs the expression of a gene in Sca-1 + cells, said therapeutic gene being under the control of said promoter that directs the expression in Sca-1 + cells, together with, optionally, one or more pharmaceutically acceptable excipients .
• Said DNA construct comprising the promoter and the therapeutic gene can be obtained by conventional genetic engineering techniques. Excipients that may be present in the pharmaceutical composition of the invention will depend, among other things, on the manner of administration of said pharmaceutical composition. A review of the different forms of administration of active ingredients, the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faul ⁇ i Trillo, Luzán 5, SA de Ediations, 1993.
• the active ingredient (DNA construction) of the pharmaceutical composition of the invention comprises vectors or systems that help the process of transferring an exogenous gene to a cell, which contain inside said DNA construct, which can be viral, for example, based on adenovirus, lentiviras, retrovims, etc., or non-viral such as the DNA-liposome, DNA-polymer, DNA-polymer complexes -liposome, etc. [see “Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)].
• the invention also relates to the use of a promoter that directs the expression of a gene in Sca-1 cells in the preparation of a pharmaceutical composition for the treatment and / or prevention of a chromosomal abnormality associated with human pathology that has its origin in stem cells, for example, chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of line B, acute lymphoblastic leukemia of line T, or with the migration of hematopoietic or embryonic stem cells (for example, alterations in the c receptor -kit or its ligand).
• a promoter that directs the expression of a gene in Sca-1 cells in the preparation of a pharmaceutical composition for the treatment and / or prevention of a chromosomal abnormality associated with human pathology that has its origin in stem cells, for example, chromosomal abnormalities associated with chronic myeloid leukemia, acute lymphoblastic leukemia of line B, acute lymphoblastic leukemia of line T, or with the migration
• Example 1 describes the generation of transgenic mice
• Example 2 demonstrates the utility of said transgenic mice as models for the in vivo study of human pathologies originating from stem cells.
• the characteristic chromosomal translocations that lead to gene fusions encoding chimeric proteins associated with human leukemia have been selected (see Example 2).
• the altered expression of said gene fusions has been implicated in a characteristic subgroup of human leukemias.
• Transgenes containing said gene fusions have been introduced into mouse genomes in which the expression of said transgenes is satisfactorily directed by the mouse pLy-6E.l promoter in Sca-1 cells.
• the pLy-6E.l promoter was used to direct tissue specific expression of the different transgenes (cDNA) in C57BL / 6 x CBA mice (Jackson Laboratory). This promoter is well characterized and the 16 kilobase (kb) fragment used contains all the elements necessary for selective expression in Sca-1 + cells [Miles C, Sánchez MJ, Sinclair A, and Dzierzak, E (1997). "Expression of the Ly-6E.l (Sca-1) transgene in adult hematopoietic stem cells and the developing mouse embryo"; Development 124, 537-547].
• genes used have been the following: BCR-ABL p210 human, gene fusion that occurs as a result of t (9; 22) (q34; ql 1) and is associated with chronic myeloid leukemia; the patients who present this chromosomal anomaly develop over time a blastic crisis, which is an evolutionary phenomenon characteristic of said disease;
• BCR-ABL p190 human an oncogene generated by the chromosomal translocation t (9; 22) and associated with acute lymphoblastic leukemia of line B;
• Murine Slug a gene that participates in the mobilization of hematopoietic stem cells
• Murine Snail a gene from the Slug family that participates in the migration of embryonic stem cells
• Human HOX11 a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of T-line
• Human RHOM2 / LMO-2 a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of T-strain
• Y a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of T-strain
• Murine TALl a gene activated by chromosomal abnormalities associated with acute lymphoblastic leukemia of strain T.
• Activatable genes identified as BCR-ABL p21 °, BCR-ABL 190 , HOX11, RHOM2 / LMO-2 and TALl are described in Annu. Rev. Genet. (1997) 31: 429-453; the Slug gene is described in Science (1994) 264: 835-849; and the Snail gene is described in Developmental Biology (1998) 198: 277-285; Development (1998) 125: 3111-3121; and Gene (2000) 257: 1-12.
• mice The mice used are C57BL / 6 x CBA and the mothers are CD1; These animals can be purchased commercially, for example, from The Jackson Laboratory (USA).
• the different cDNAs (human BCR-ABL p190, human BCR-ABL 210 , murine Slug, murine Snail, human HOX11, human LMO2 / RHOM2 and murine TALl) were cloned into the Clal site of the mouse pLy-6E.l promoter.
• the different cDNAs were obtained by digestion with the appropriate restriction endonucleases and each cDNA was cloned into the vector containing the pLy-6E.l promoter digested with Clal by conventional techniques [Molecular Cloning, thrid edition, CSHL Press by Sambrook and Russell, 2001].
• ABL p190 , Sca-1 + Slug, Sca-1 + Snail and Sca-l + RHOM2 / LMO-2 obtained a linear DNA fragment for microinjection by Notl digestion, while for the generation of the Sca-1 transgenic mice + HOXl ly Sca-1 + TALl
• the linear DNA fragment for microinjection was obtained by digestion with BamHI.
• the different linear DNA fragments were microinjected into fertilized oocytes of C57BL / 6J x CBA mice.
• the founding transgenic mice were identified by Southern blot analysis using specific probes that recognized said cDNAs, in DNA samples extracted from the tails of mice.
• the preparation of the fertilized oocytes, the microinjection of the DNA constructs containing the operably linked promoter and gene, the reimplantation of the fertilized oocytes to which said DNA constructs had been injected into the pseudopregnated mother mothers and the maintenance of Nurse mothers during pregnancy were performed using conventional techniques [Hogan, Constantini & Lacy (1986), "Manipulating the Mouse Embryo.
• the progeny / offspring of the transgenic mice was obtained by crossing the founder mouse with C57BL / 6 x CBA mice and identifying the positives by Southern blot analysis with specific probes that recognized the cDNAs.
• CD45R / B220 For staining for cytometry the following anti-mouse monoclonal antibodies conjugated to phycoerythrin (PE) (all of Pharmingen) were used: CD45R / B220, Thy-1.1 and Thy-1.2, myeloid markers (Mac 1 / CDl Ib and Gr- 1) and Sig.
• Cell suspensions from whole blood samples obtained by routine techniques were incubated with purified anti-mouse CD32 / CD16 (Pharmingen) to block receptor binding via Fe and with appropriate dilution of the different antibodies to room temperature or 4 ° C, respectively. Erythrocytes were lysed using lysis solution (Becton Dickinson).
• MRNA was obtained from different chimeric mouse tissues. By reverse transcription of each RNA preparation treated with DNase I (HT) of RNase cDNA was obtained. The cDNA was subjected to PCR using specific primers in each case [the direct initiator (5 ') corresponded to the first 20 bases of the cDNA and the reverse initiator (3') was complementary to the last 20 bases of the cDNA]. The reactions were carried out following the instructions of the supplier of Taq polymerase (Perkin-Elmer Cetus) under the following conditions: 1 minute at 95 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C for 25 cycles with one final elongation of 10 minutes at 72 ° C.
• Taq polymerase Perkin-Elmer Cetus
• Tissue samples were fixed in 4% formaldehyde in PBS and embedded in paraffin. Thin sheets were cut and processed and stained with hematoxylin-eosin by routine techniques. The plates were examined and photographed.
• DNA from various tissues was prepared by conventional techniques.
• the DNA was digested with BamHI and the Southern blots were analyzed with a probe of a specific imnunoglobulin [Blood (rapid publication) 90: 2168-2174 (1997)].
• Transgene expression was observed in both lines and the progeny multiplied to level F7 (Generation 7). Transgene expression was demonstrated by PCR and / or Western blot analysis. Both cell lines showed preferential expression in Sca-1 + cells. Transgene expression was detected in both male and female mice, similar findings being found in both lines of the Sca-l + BCR-ABL 210 transgenic mice.
• Sca-1 + Snail produce embryonic stem cell migration but not leukemia
• Sca-1 + HOXll develop acute lymphoblastic leukemias of strain T
• Sca-1 + TAL1 develop acute lymphoblastic leukemias of strain T. In all cases the expression of the transgene was observed in both lines and the progeny multiplied to level F7 (Generation 7). Transgene expression was demonstrated by PCR and / or Western blot analysis. Both cell lines showed preferential expression in Sca-1 cells. Transgene expression was detected in both male and female mice, similar findings being found in both lines of the transgenic mice.
• EXAMPLE 2 Production of leukemia in transgenic mice Although in human pathology the chimeric products of the genes BCR-ABL p210 , BCR-ABL 190 , Sca-1 + HOXl l, Sca-l + RHOM2 / LMO-2 and Sca-1 + TALl , are associated with different types of leukemia, namely chronic myeloid leukemia (BCR-ABL p21 °), acute lymphoblastic leukemia of line B (BCR-ABL pl9 °) and acute lymphoblastic leukemia of lineage T (HOX11, RHOM2 / LMO-2 and TALl), the current murine models for these leukemias have failed to consistently reproduce these pathologies [Annu. Rev. Genetics (1997) 31: 429-453; Current Genomics (2000), 1: 71-80] due to the difficulty of choosing a promoter to manipulate expression in the appropriate cell type.
• BCR-ABL p21 ° chronic myeloid leukemia
• mice 1 x 10 or " cells were injected intravenously into normal non-irradiated NOD / SCE) mice. All injected mice progressively developed leukemia within 6-11 weeks after On the contrary, none of the 20 mice injected with cells from 10 control mice developed a leukemia whose origin was attributed to the donor. The transplanted cells developed the same leukemia class, and the origin of the leukemic clones of the mice Donors were confirmed by means of a PCR analysis revealing the presence of the core transponders.
• the male and female transgenic mice showed, uniformly, the same symptoms (of the corresponding pathology) beginning at 8 weeks of age and increasing the clinical signs over time to death. 100%> of the mice, which took place at 12-16 months of age.
• the transgenic founding mice were male and female and developed the clinical symptoms of the corresponding disease, similar to that of the other transgenic mice. All transgenic mice died from tumors at 14-18 months of age. The percentage of survival in the lines of the 2 founding mice was similar in each case. No tumors were observed in control groups consisting of an equal number of non-transgenic bait mice. Often the animals suffered tachypnea so they were sacrificed.
• the murine models studied not only resemble the regrouping that takes place in human leukemias considered [chronic myeloid leukemia, acute lymphoblastic leukemia of strain B and acute lymphoblastic leukemia of strain T] but also reproduce the same phenotype with which the gene fusions involved are associated in human pathologies.
• These results validate these murine models as ideal models for the in vivo study of the biology of the transgenes tested (BCR-ABL p21 °, BCR-ABL p190 , Sca-1 + HOXll, Sca-l + RHOM2 / LMO-2 and Sca - 1 + SIZ1).

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